Patient prognosis rapidly deteriorates after the detection of distant metastasis. During the metastatic cascade, host-cancer interactions signal the development of highly localized ?metastatic niches? in remote tissues that suppress normal immune function. Natural killer (NK) cells within remote organs normally eliminate cancer cells through broad innate and adaptive mechanisms, yet they fail to do so at the metastatic niche. While NK cell-based immunotherapy has recently had clinical success against hematopoietic cancer, strategies for improving NK cell function within the microenvironment of the metastatic niche are required to improve outcomes for patients with solid cancers. However, the transient and a priori unknown highly localized nature of the metastatic niche within bulk organs makes detailed dynamic study of NK cell function challenging. Circumventing this limitation, the Shea lab has developed a biomaterial implant that recruits metastatic cancer cells in vivo, and by explanting tissue from the implant into a vascularized 3D microfluidic device can create an integrated in vivo and in vitro synthetic model of the metastatic niche. Dynamic systems analysis of the integrated in vivo and in vitro synthetic metastatic niche will be used to discover and test signaling targets for NK cell-based immunotherapy at the metastatic niche. Aim 1 will investigate the hypothesis that the local soluble microenvironment of the early metastatic niche prevents efficient homing of activated NK cells. The dynamic changes in the secretome of the synthetic metastatic niche and the trafficking of NK cell subtypes will be characterized. Next, specific candidate soluble factors that alter NK cell behavior within the context of the synthetic metastatic niche will be studied to identify the driving pathways by RNA sequencing and parallelized live cell dynamic reporters. Aim 2 will test the hypothesis that pathways active in ?serial killing? NK cells can serve as therapeutic targets and attempt to improve NK cell activation at the metastatic niche for therapy. A live single cell dynamic pathway discovery technology will be applied to individual NK cells as they enter a dynamic serial killing state to investigate how serial killing pathways are altered by the microenvironment of the in vitro metastatic niche. Next, the in vitro metastatic niche will be used to screen strategies for rescuing NK cell cytotoxicity at the metastatic niche including cytokine- induced memory, galectin-1 inhibition, TGF-? signaling blockade, and mechanisms revealed by Aims 1 and 2. Successful strategies will be validated in vivo and continued into survival studies to test if they extend the survival advantage provided by the current metastatic niche implant. Taken together these studies will identify and test targets for therapeutic intervention in NK cell-based and NK cell adjuvant immunotherapies against metastatic solid cancers.